Inhibition of interaction of PSD93 and PSD95 with nNOS and NMDA receptors

ABSTRACT

PSD-95/SAP90 antisense-treated animals not only experience a significant decrease in MAC for isoflurane, but also experience an attenuation in the NMDA-induced increase in isoflurane MAC. PSD-95/SAP90 appears to mediate the role of the NMDA receptor in determining the MAC of inhalational anesthetics. Suppression of the expression of PSD-95/SAP90 in the spinal cord significantly attenuates responses to painful stimuli mediated through the N-methyl-D-aspartate receptor activation. In spinal cord neurons PSD-95/SAP90 interacts with the N-methyl-D-aspartate receptor subunits 2A/2B. Activation of the N-methyl-D-aspartate receptor in spinal hyperalgesia results in association of the N-methyl-D-aspartate receptor with PSD-95/SAP90. PSD-95/SAP90 is required for hyperalgesia triggered via the N-methyl-D-aspartate receptor at the spinal cord level.

[0001] This application claims the benefit of provisional applicationsSerial No. 60/242,580 filed Oct. 23, 2000, and Ser. No. 60/203,894 filedMay 12, 2000, the entire contents of which are expressly incorporatedherein.

[0002] This invention was made using funds from the U.S. governmentunder grants from the National Institutes of Health numbered RO GM49111and RO1 HL39706. The U.S. government therefore retains certain rights inthe invention.

BACKGROUND OF THE INVENTION

[0003] The potency of anesthetic agents to inhibit the ability of apatient to respond with movement to painful stimuli has long been usedas a test of anesthetic action. This potency, characterized by its ED₅₀,is widely known as the minimum alveolar concentration (MAC). Severallines of evidence have shown that spinal NMDA receptor activation mightplay a key role in the processing of nociceptive information1,29-30 andin the determination of the MAC of inhalational anesthetics.³¹⁻³³ Forexample, the NMDA receptors are distributed mainly in the superficiallaminae of the spinal cord.^(12,28) Both repetitive C-fiber stimulationand direct application of glutamate or NMDA produce spinal neuronalsensitization and enhance responsiveness, which can be blocked by NMDAreceptor antagonists. Behavioral studies demonstrate that spinaladministration of NMDA produces thermal hyperalgesia, caudally directedscratching and biting, and exaggerated responsiveness to lighttouch.^(8,37-39) Moreover, antagonism of the spinal NMDA receptorsproduces antinociception in numerous animal models of pain³⁹⁻⁴⁴ andreduction in the MAC of isoflurane.³¹⁻³³ However, the molecularmechanisms underlying these actions remain unknown.

[0004] The postsynaptic density (PSD), a highly organized cytoskeletalstructure found adjacent to the postsynaptic membrane of excitatorysynapses, is believed to play a role in the organization of receptorsand related proteins involved in synaptic signaling.⁴⁵⁻⁵⁵ A number ofproteins enriched in the PSD have been characterized.⁴⁷⁻⁴⁸ One of theseproteins, postsynaptic density-95 (PSD-95)/synapse-associated protein-90(SAP90), is an abundant scaffolding molecule that binds and clusters theNMDA receptor preferentially at synapses in the brain and spinal cord.^(3,4,5,6,7,9,49) This raises the possibility that PSD-95/SAP90 might beinvolved in many physiological and pathophysiologic actions triggeredvia the NMDA and perhaps other receptors in the central nervous system.Indeed, suppression of PSD-95/SAP90 expression attenuated excitotoxicityproduced via NMDA receptor activity in brain neurons.²³ The lack ofPSD-95/SAP90 revealed an enhanced NMDA-dependent long-term potentiationand impaired learning.¹⁶

[0005] The role of the N-methyl-D-aspartate (NMDA) receptor in spinalhyperalgesia has been demonstrated by behavioral, electrophysiologicaland neurochemical findings.^(1,8,21,26) However, the molecularmechanisms underlying these actions are unclear. The NMDA receptorconsists of two distinct types of subunits: NMDAR1 (NR1) and NMDAR2A-D(NR2A-D).¹⁹ The C-termini of the NR2 subunits interact with PSD-95/SAP90and other members of the membrane-associated guanylate kinase (MAGUK)family in the brain.^(2,6,9,10,17,20) This raises the possibility thatthe sensory hyperalgesia produced through NMDA receptor activation isdetermined by NMDA receptor-bound proteins of the MAGUK family in thespinal cord.

[0006] There is a need in the art for new ways of treating andpreventing hyperalgesia and chronic and acute pain. In addition, thereis a need in the art for new and safer ways of rendering patientsunconscious via general anesthesia or by sedating them.

SUMMARY OF THE INVENTION

[0007] One embodiment of the invention provides a method for relievingacute or chronic pain. According to the method an effective amount of anagent which inhibits expression of PSD93 or PSD95 is administered to asubject in need of pain relief. The agent relieves acute or chronic painexperienced by the subject.

[0008] Another embodiment of the invention provides a method fortreating or preventing hyperalgesia. According to the method aneffective amount of an agent which inhibits expression of PSD93 or PSD95is administered to a subject who has or is at risk of developinghyperalgesia. The administration relieves or prevents hyperlagesiaexperienced by the subject.

[0009] Another aspect of the invention is a method of reducing athreshold for anesthesia. An anesthetic and an agent which inhibitsexpression of PSD93 or PSD95 are administered to a subject. The amountof anesthetic administered achieves a desired anesthetic effect eventhough the amount administered is less than the amount required in theabsence of the agent to achieve the desired anesthetic effect. Thisminimizes the serious side effects of the anesthetics includingcardovascular and respiratory depression.

[0010] The present invention also provides an isolated and purifiedantisense polynucleotide which is complementary to PSD95 or PSD93 mRNA.

[0011] Another embodiment of the invention is a method for relievingacute or chronic pain. An effective amount of an agent which inhibitsinteraction of a first protein selected from the group consisting ofPSD93 and PSD95, with a second protein selected from the groupconsisting of nNOS and NMDA receptor, is administered to a subject inneed thereof. The agent does not cause cardiovascular or respiratorydepression. The administration relieves acute or chronic painexperienced by the subject.

[0012] Also provided is an alternative method for treating or preventinghyperalgesia. An effective amount of an agent which inhibits interactionof a first protein selected from the group consisting of PSD93 andPSD95, with a second protein selected from the group consisting of nNOSand NMDA receptor, is administered to a patient experiencinghyperalgesia or who is at risk of developing hyperalgesia. The agentdoes not cause cardiovascular or respiratory depression. Hyperalgesiaexperienced by the subject is relieved or prevented by theadministration.

[0013] Also provided by the present invention is a method of reducing athreshold for anesthesia. An anesthetic and an agent which inhibitsinteraction of a first protein selected from the group consisting ofPSD93 and PSD95, with a second protein selected from the groupconsisting of nNOS and NMDA receptor, are administered to a subject. Theagent does not cause cardiovascular or respiratory depression. Theamount of anesthetic administered is less than the amount required inthe absence of the agent to achieve a desired anesthetic effect. Thedesired anesthetic effect is thus achieved.

[0014] The present invention also provides a method of anesthetizing asubject. An agent which inhibits expression of PSD93 or PSD95 isadministered to a subject. The agent renders the subject unconscious orsedated.

[0015] Another embodiment of the invention provides a method ofanesthetizing or sedating a subject. An agent which inhibits interactionof a first protein selected from the group consisting of PSD93 andPSD95, with a second protein selected from the group consisting of nNOSand NMDA receptor, is administered to a patient. The agent does notcause cardiovascular or respiratory depression. The agent renders thesubject unconscious or sedated.

[0016] Yet another aspect of the invention is a method of screening forsubstances useful for relieving pain or inducing unconsciousness orsedation. A test substance is contacted with a first protein and asecond protein under conditions where the first protein and the secondprotein bind to each other. The first protein is selected from the groupconsisting of PSD93, PSD95, and a combination thereof. The secondprotein is selected from the group consisting of nNOS, NMDA receptor,NR2A subunit, NR2B subunit, and combinations thereof. The mixture ofproteins is assayed to determine the binding of the first protein to thesecond protein. Any parameter which reflects that binding can beassayed. Such parameters include the amount of free nNOS, the amount offree PSD93, the amount of free PSD95, the amount of free NMDA receptor,the amount of free NR2A subunit, the amount of free NR2B subunit, theamount of bound NNOS, the amount of bound PSD93, the amount of boundPSD95, the amount of bound NMDA receptor, the amount of bound NR2Asubunit, the amount of bound NR2B subunit and combinations of them. Atest substance which increases the amount of free nNOS, free PSD93, freePSD95, free NMDA receptor, free NR2A subunit, or free NR2B subunit, orwhich decreases the amount of bound nNOS, bound PSD93, bound PSD95,bound NMDA receptor, bound NR2A subunit, or bound NR2B subunit isidentified as a candidate drug for relieving pain or inducingunconsciousness or sedation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1A and 1B. Expression of PSD-95/SAP90 mRNA and protein in thespinal cord. In FIG. 1Aa, immunoblot showing the expression ofPSD-95/SAP90 in the PSD fractions of the spinal cord (SC), dorsal rootganglion (DRG) and other brain regions (as positive controls) in thenormal rats. HI: hippocampus; CO: cortex; CE: cerebellum. In FIG. 1Ab,immunoblot showing representative effects of PSD-95/SAP90 antisense(AS), missense (MS) and sense (SE) ONDs, as well as saline (SA), on theexpression of PSD-95/SAP90, nNOS and NR2A/2B in the spinal cord. PC:positive control tissue from hippocampus. Asterisk: non-specific band bythe secondary antibody, useful to control for protein loading and blotexposure times. In FIG. 1B, RT-PCR analysis showed that 0.737 Kb mRNAwas detected in the spinal cord and other brain regions (hippocampus,cortex, cerebellum and brainstem), but not in muscle. PCR product wasdirectly cloned into the TA cloning vector and verified as PSD-95/SAP90by automatic DNA sequencing. β-actin mRNA was used as a loading control.

[0018]FIG. 2A and 2B. Distribution of PSD-95/SAP90 immunoreactivity inlumbar enlargement segments of the spinal cord. The PSD-95/SAP90immunoreactivity was localized mainly in lamina I and outer lamina II(A). Under high magnification, many PSD-95/SAP90 immunoreactive punctawere observed (B). Scale bars: 200 μM in A; 40 μm in B.

[0019]FIG. 3. Identification of a ternary complex assembled byPSD-95/SAP90 with NR2A/2B and nNOS in the spinal cord neurons.PSD-95/SAP90 antibody immunoprecipitated not only PSD-95/SAP90 but alsoNR2A/2B and nNOS. In contrast, endothelial NOS (eNOS) was notimmunoprecipitated by PSD-95/SAP90 antibody. Ten μg protein was loadedin INPUT lane and 100 μg in other lanes.

[0020]FIG. 4. Effect of intrathecal administration of NMDA on isofluraneMAC in the saline- and PSD-95/SAP90 antisense ODN-treated groups. Dataare presented as mean ±SD. n=5 animals for each group, except n=14 forthe saline-treated (control) group. ** Significantly different fromcontrol (P<0.01).

DETAILED DESCRIPTION OF THE INVENTION

[0021] It is a discovery of the present inventors that PSD95 and PSD93mediate the interaction of NMDA receptors and nNOS in the spinal cord,and are involved in generating responses to painful stimuli. Theinventors have found that inhibition of the interaction of NMDAreceptors and nNOS via PSD95 and PSD93 can attenuate responses topainful stimuli, as well as lower thresholds for anesthetics. Moreover,we have found that inhalational anesthetics themselves inhibit theinteraction of NMDA receptors and nNOS via PSD95 and PSD93. Thus new andimproved anesthetics and sedatives can be identified using theidentified interaction as an assay system.

[0022] Acute or chronic pain can be relieved or prevented according tothe present invention by administering to a subject an effective amountof an agent which inhibits expression of PSD93 or PSD95. The agents ofthe present invention also can be used to treat or prevent hyperalgesia,as well as to reduce a threshold for anesthesia. The agent used can bean antisense oligonucleotide (ODN) which is complementary to mRNAencoding PSD93 or PSD95. Preferably the antisense oligonucleotide iscomplementary to nucleotides encoding a PDZ domain. More preferably theantisense oligonucleotide is complementary to nucleotides 241 to 258 ofPSD95. Any agent which acts to specifically inhibit transcription ortranslation of PSD93 or PSD95 can be used. Oligonucleotides useful inthe invention can be naked oligonucleotides or can be administered in avector, liposome, particle or other protective formulation. If in avector, the vector can express RNA molecules which are complementary tothe native PSD93 or PSD95 mRNAs. Also encompassed by the presentinvention are oligonucleotides which contain nucleotide analoguemoieties to render the oligonucleotides less susceptible to enzymaticdegradation. Suitable nucleotide analogue moieties are known in the artand include phophorothioates.

[0023] Agents according to the present invention can be administered anyway known in the art which is convenient and efficient for theparticular agent and the application. Preferably the agent isadministered intrathecally, per os, or intravenously. However, othermeans can be used as appropriate, including subdermal, subcutaneous,rectal, intraperitoneal, subarachnoid, caudal, epidural, inhalational,and intramuscular administrations. Anesthetics and sedatives used in themethods of the present invention can also be administered by any ofthese same means. Preferred anesthetics according to the invention areinhalational anesthetics, including halothane, isoflurane, desflurane,xenon, and sevoflurane.

[0024] Compositions are provided for inhibiting expression of PSD95 orPSD93. Such compositions comprises an isolated and purified antisensepolynucleotide which is complementary to PSD95 or PSD93 mRNA. Preferablythe polynucleotide is complementary to nucleotides encoding a PDZdomain. Any of the three such domains can be targeted, although thethird such domain, i.e., the C-terminal PDZ domain, may be the mosteffective. One particular oligonucleotide which has been found to beeffective is complementary to nucleotides 241 to 258 of PSD95. Theanalogous nucleotides of PSD93 can also be used. The polynucleotide canbe formulated in a pharmaceutically acceptable vehicle so that it can beused to prevent pain or to lower an anesthetic or sedative threshold.Particular vehicles which are suitable for intrathecal or inhalationaltherapy can be advantageously used. The formulations can be in liquid orvapor form. They can be vaporized by bubbling a gas through them.Preferably the formulations of the invention will be manufactured underregulatory-approved conditions for administration to humans.Requirements for such formulations typically include sterility andfreedom from pyrogens.

[0025] Not only can agents which specifically inhibit the expression ofPSD93 or PSD95 be used in the methods of the present invention, but alsoagents which inhibit the interaction of PDS93 or PSD95 with either nNOSor NMDA receptors. Such agents can be used for the same purposes asdiscussed above, for relieving acute or chronic pain, for reducing thethreshold for anesthetics and sedatives, and for anesthetizing andsedating patients directly. Agents useful according to the presentinvention do not cause cardiovascular or respiratory depression. Suchagents can be administered to the same populations of patients asdiscussed above, i.e., those in need of anesthesia, those in need ofrelief from chronic or acute pain, and those who experience hyperalgesiaor are at risk of developing hyperalgesia. Such patients include thosewhose pain is mechanical, thermal, neuropathic, or inflammatory inorigin.

[0026] Protein interaction-inhibitory agents of the invention preferablybind to a PDZ domain of any of the binding participants, including nNOS,NMDA receptors, PDS93 or PDS95. Typically and preferably the agent doesnot impair motor function, i.e.,locomotion. Such agents can beidentified by any of a number of screening techniques which rely on theinhibition of expression or interactions of PDS93 or PDS95. Generally,test substances are contacted with a first protein and a second proteinunder conditions where the first protein and the second protein bind toeach other. The first protein is PSD93, PSD95, or a combination the twoproteins. The second protein can be NNOS, NMDA receptor, NR2A subunit,NR2B subunit, or combinations of these proteins. Fusion proteins whichcontain all or relevant binding portions of these proteins can be used,as is desirable for ease of detectability or purification and handling.The amount of protein which is bound or free in the presence and absenceof the test substance can be determined by any techniques known in theart. Test substances which increase the amount of free binding partnersor which decrease the amount of bound binding partners are identified ascandidate drugs for relieving pain or inducing unconsciousness orsedation.

[0027] Many protein-protein binding assays are known in the art and anysuch format or technique can be used as is convenient. In some assaysthe proteins are contacted in vitro. In other assays the proteins are inyeast cells containing recombinant forms of the first and secondproteins, and the test substance is contacted with the whole yeastcells. Such assays include the well-known two hybrid assays, in whichbinding of two binding partners reconstitutes a transcriptionalactivating activity. In these assays the first and second bindingpartners are each fused to a first and second yeast protein whichreconstitute a functional transcriptional activator when brought intophysical proximity by binding of the first recombinant protein to thesecond recombinant protein. Colorimetric, enzymatic, or growth assayscan be used to determine the transcriptional activation reconstitution.Candidates which are identified as having inhibitory activity in suchassays can be further tested in an animal to determine if the candidatedrug relieves pain or induces unconsciousness or sedation.

[0028] PSD-95/SAP90 antisense-treated animals not only experience asignificant decrease in MAC for isoflurane, but also experience anattenuation in the NMDA-induced increase in isoflurane MAC. PSD-95/SAP90appears to mediate the role of the NMDA receptor in determining the MACof inhalational anesthetics. Suppression of the expression ofPSD-95/SAP90 in the spinal cord significantly attenuates responses topainful stimuli mediated through the N-methyl-D-aspartate receptoractivation. In spinal cord neurons PSD-95/SAP90 interacts with theN-methyl-D-aspartate receptor subunits 2A/2B. Activation of theN-methyl-D-aspartate receptor in spinal hyperalgesia results inassociation of the N-methyl-D-aspartate receptor with PSD-95/SAP90.PSD-95/SAP90 is required for hyperalgesia triggered via theN-methyl-D-aspartate receptor at the spinal cord level.

[0029] The pretreatment of PSD-95/SAP90 antisense ODN but not sense ormissense ODN produced a remarkable reduction in isoflurane MAC. This wasnot accompanied by changes in ether blood pressure or heart rate.Furthermore, the PSD-95/SAP90 antisense ODN blocked NMDA-inducedincrease in isoflurane MAC. The deficiency of PSD-95/SAP90 expressionmay produce anesthetic and analgesic actions at the spinal cord leveland PSD-95/SAP90 might mediate the role of the NMDA receptor indetermining the MAC of inhalational anesthetics.

[0030] Antisense ODNs have been widely used as research tools, and evenas drugs in clinical trials. Antisense ODNs inhibit protein expressionby the mechanisms of (1) steric blockade of ribosomal subunit attachmentto mRNA at the 5′ cap site; (2) interference with proper mRNA splicingthrough antisense binding to splice donor or splice acceptor sites; (3)RNase-H-mediated degradation of hybridized mRNA.¹⁸ The proper design andcontrols of experiments are critical in demonstrating a true antisenseeffect. The specificity of intrathecal treatment with PSD-95/SAP90antisense ODN has been shown. First, we designed the standard controlsof equivalent sense sequence and missense ODNs. Neither had any effecton the isoflurane MAC. This indicates the specificity of the inhibitionobserved with the antisense ODN. Second, all of the ODNs had beensearched to exclude non-specificity of the sense or antisense ODNs andto show that missense ODN did not match any confounding sequences in theGenBank database. Moreover, our previous results have demonstrated thatantisense ODNs only suppressed the expression of PSD-95/SAP90 but notthe expression of NMDA receptor subunits NR2A/2B, neuronal nitric oxidesynthase or SAP102 (a protein that is closely related to the targetedprotein) in the spinal cord.⁴⁹ The effects observed following treatmentwith the PSD-95/SAP90 antisense ODN are unlikely to be explained bychanges in the expression of other proteins. Finally, the antisense ODNsat the doses used only affected isoflurane MAC without untoward effectsin any of the treated animals including the antisense groups.Considering these several lines of evidence, we believe that the effectswe have described may be due to a direct and selective interference ofthe antisense ODN with mRNA transcripts of PSD-95/SAP90 and to theblockade of protein production via binding to the nucleotides ofPSD-95/SAP90 mRNA.

[0031] The regional expression and function of PSD-95/SAP90 in themammalian brain have been investigated using a variety of experimentalapproaches.^(3,45,7,9) PSD-95/SAP90 immunoreactivity was found mainly incortex, hippocampus and cerebellum.⁵⁴⁻⁵⁶ In brain neurons, suppressionof PSD-95/SAP90 expression that selectively disrupted physical linkageof the NMDA receptor with neuronal nitric oxide synthase has beendemonstrated to attenuate excitotoxicity and Ca2+-activated nitric oxideproduction via NMDA receptor activity.²³ Mice carrying a targetedmutation in the PSD-95/SAP90 gene showed an enhanced NMDA-dependentlong-term potentiation and impaired learning.¹⁶ Recently, we found thatPSD-95/SAP90's mRNA and protein also were enriched in the spinal cordand selectively distributed in the superficial dorsal horn, wherePSD-95/SAP90 expression overlapped with that of the NMDAreceptor.^(12,28) In the spinal neurons, PSD-95/SAP90 interacted withthe NMDA receptor subunits 2A/2B.⁴⁹ Behavioral studies showed thatintrathecal administration of antisense ODN for PSD-95/SAP90significantly attenuated facilitation of the tail-flick reflex triggeredthrough the NMDA receptor activation.⁴⁹ The evidence above indicatesthat activation of the NMDA receptor in spinal hyperalgesia results inassociation of the NMDA receptor with PSD-95/SAP90 and that PSD-95/SAP90is required for the spinal mechanisms of hyperalgesia. This suggeststhat PSD-95/SAP90 may be involved in the processing of pain and thatdeficiency of PSD-95/SAP90 may produce analgesic action at the spinalcord level. Such an action is consistent with the effect of thedeficiency of PSD-95/SAP90 on MAC. Doses of antisense ODNs did not causemotor and general behavioral dysfunction when administered intrathecallyin rats. The effect of suppression of spinal PSD-95/SAP90 expressionthat resulted in the reduction in MAC may be due to effects on analgesiaalone. However, PSD-95/SAP90 has been demonstrated to be involved in themechanisms of long-term potentiation and learning.¹⁶ An effect ofantisense ODN on righting reflex was not observed. The possibility ofthese actions of the antisense ODNs in the central nervous system couldnot be ruled out from the current study since the intrathecal antisenseeffect had a segmental nature.

[0032] A role for the NMDA receptors in determining the MAC ofinhalational anesthetics is suggested by the fact that the systemic orintrathecal administration of NMDA antagonists significantly reduces theMAC of isoflurane in rats, which is completely reversed to control levelby intrathecal administration of NMDA.³¹⁻³³ The current study irtherindicated that intrathecal administration of NMDA increased the MAC ofisoflurane in saline-treated rats. Interestingly, in antisenseODN-treated rats, intrathecal injection of NMDA did not affect the MACof isoflurane. PSD-95/SAP90 localization completely overlapped with theNMDA receptor subunits 2A12B in spinal superficial dorsal horn.⁴⁹Furthermore, the PSD-95/SAP90 antibody was able to immunoprecipitate notonly PSD-95/SAP90 but also NR2A/2B in vivo.⁴⁹ These findings demonstratethat PSD-95/SAP90 interacts with NR2A/2B in the spinal cord in vivo.Combined with the current results, it is suggested that PSD-95/SAP90 isessential for the actions of the NMDA receptor in determining the MAC ofinhalational anesthetics.

[0033] In our experiments, no significant hemodynamic effects wereobserved in the ODN-treated animals during isoflurane anesthesia.However, intrathecal administration of NMDA resulted in a significantincrease in systolic and diastolic blood pressure during isofluraneanesthesia in both the saline- and antisense ODN-treated groups. It hasbeen demonstrated that sympathetic preganglionic neurons located in theintermediate nucleus of the spinal cord are integral elements in theneural pathway linking the central nervous system to sympathetic nervessupplying the heart and blood vessels.^(57,58) The effects of NMDA onblood pressure may be due to the involvement of the NMDA receptor inregulation of sympathetic output at the spinal cord level. Inimmunohistochemical studies, glutamate and its receptors were found inthe intermediolateral nucleus of the thoracic spinal cord.^(59,60)Intrathecal administration of NMDA at the T10 level increased arterial61,62 pressure. This action was blocked by NMDA receptorantagonists.^(61,62) It is likely that NMDA, administered intrathecallyat the lumbar level, activates spinal sympathetic activity in theintermediolateral nucleus and produces the increase in blood pressure.The antisense ODNs had no effect on the hemodynamics or on theNMDA-induced increase in blood pressure, a finding which is consistentwith our previous observation that PSD-95/SAP90 was absent or present atextremely low levels in the intermediolateral nucleus of the spinalcord.⁴⁹ It could be that the second message signaling pathways in thesomatic and the sympathetic nervous systems are different with respectto the NMDA receptor. Hong et al ⁶¹ and West et al ⁶² reported thatmicroinjection of NMDA into the intermediolateral nucleus at the spinalT₂ level or intrathecal injection of NMDA at the T₁₀ level produced anincrease in heart rate. Interestingly, the effect of NMDA on heart ratewas not observed in either saline- or antisense ODN-treated groups inthe present study. The reason for this discrepancy between the previousand the present studies is not clear and may be due to a difference inanesthetic agents (isoflurane in the present study vs urethane, chloralhydrate and sodium pentobarbitone). It is interesting to note thatintrathecal administration of the NMDA receptor antagonist, APV,produced a dose-related decrease in arterial pressure but not in heartrate.^(61,63) These data suggest that there is a tonic activation of theNMDA receptor in the spinal sympathetic pathway to the vessels but notto the heart.

[0034] MAC for isoflurane was significantly decreased and theNMDA-induced increase in isoflurane MAC was attenuated in thePSD-95/SAP90 antisense-treated animals. The binding of PSD-95/SAP90 tothe NMDA receptor preferentially at synapses in the spinal cord andbrain suggests that PSD-95/SAP90 may mediate the role of the NMDAreceptor in determining the MAC of inhalational anesthetics.

EXAMPLES Example 1

[0035] This example demonstrates that PSD-95 is necessary for thermalhyperalgesia.

[0036] To examine whether PSD-95/SAP90 was required for thermalhyperalgesia triggered through NMDA receptor activation, we made anantisense oligonucletide (OND) corresponding to the PDZ domainnucleotides 241 to 258 (5′-TGTGATCTCCTCATACTC-3′; SEQ ID NO: 1) of ratPSD-95/SAP90 MRNA, as well as the sense OND and missense OND(5′-AAGCCCTTGTTCCCATTT-3′; SEQ ID NO: 2). All of the ONDs were comparedto the Gene Bank database (GenBank accession number M96853) and foundnot to be complementary to any registered nucleotide sequences. Theeffects of antisense, sense and missense ONDs on baseline andNMDA-induced tail-flick latencies were assessed. Consistent withprevious studies,^(14,15,24,26) intrathecal administration of NMDA at 5nM /10 μl (n=6) (data not shown) or 10 nM/10 μl (n=12) induced afacilitation of the tail-flick reflex (The baseline tail-flick latencywas reduced from 6.5±0.57 to 4.88±0.41 seconds.p<0.01) (Table 1). Wefound that the NMDA-produced facilitation of the tail-flick reflex wasattenuated in rats pretreated with antisense ONDs (25 μg /10 μl and 50μg /10 μl every 24 h for 4 days; n=6 each group) but not in thosepretreated with sense OND (50 μg /10 μl every 24 h for 4 days; n×6) ormissense OND (50 μg/10 μl every 24 h for 4 days; n=6) (Table 1).Antisense OND given intrathecally at 25 and 50 μg dramatically preventedthe NMDA-induced decrease of the tail-flick latency by 55% (p<0.05) and82% (p<0.01), respectively. When these rats treated with antisense ONDwere allowed to recover for an additional four days, their tail flicklatency in response to NMDA stimulation returned to normal. To identifythat NMDA-induced thermal hyperalgesia was produced specifically throughNMDA receptor activation but not non-NMDA receptor activation, weobserved the effects of a selective NMDA receptor antagonist, MK-801,and a selective non-NMDA receptor antagonist, DNQX, on NMDA-inducedfacilitation of the tail-flick reflex. As shown in Table 1, intrathecalMK-801 at 10 nM/10 μl (n=6) completely abolished facilitation of thetail-flick reflex stimulated by NMDA (p<0.01), while intrathecal DNQX at20 nM /10 μl (n=6) had no effect (p>0.05). The baseline thermal reflexis generally considered to be mediated via non-NMDA receptormechanisms.^(13-15,26) Antisense OND for PSD-95/SAP90 did not affectbaseline tail-flick latency (percentage change of TF latency was0.44±1.95) compared to the control group; Nor did sense and missenseONDs (percentage changes of TF latencies were −0.82±1.94 and −2.05±1.57,respectively). In addition, motor weakness or dysfunction was notobserved in locomotor tests (including placing reflex, grasping reflexand righting reflex) in any of the treated animals including theantisense groups (data not shown).

Example 2

[0037] This example shows that PSD-95 antisense oligonucleotide actsspecifically to inhibit PSD-95 expression.

[0038] Antisense ONDs, widely used as research tools and even as drugsin clinical trials, inhibit protein expression by the mechanisms of (1)steric blockade of ribosomal subunit attaclunent to mRNA at the 5′ capsite; (2) interference with proper mRNA splicing through antisensebinding to splice donor or splice acceptor sites; (3) Rnase-H-mediateddegradation of hybridized mRNA.¹⁸ To further examine whether the actionof antisense OND for PSD-95/SAP90 above was specifically due toselective decrease or lack of PSD-95/SAP90 but not other proteins in thespinal cord, we detected PSD-95/SAP90, NMDA receptor subunits 2A/2B(NR2A/2B), neuronal nitric oxide synthase (nNOS) and SAP-102 inhomogenates from crude lumbar enlargement segments in the normal,saline-treated (control) and OND-treated rats. PSD-95/SAP90 protein wasenriched in the postsynaptic density (PSD) fraction of the spinal cordin normal, control, sense OND- and missense OND-treated groups (FIG. 1Aa and b). In contrast, in the antisense OND-treated group, PSD-95/SAP90expression was suppressed to <15% of control (FIG. 1 Ab). No significantchange in expression of NR2A/2B, nNOS and SAP-102 was found in normal,control or OND-treated animals (FIG. 1 Ab). It is likely that theantisense OND for PSD-95/SAP90 selectively interferes with mRNAtranscription of PSD-95/SAP90 and blocks production of the protein viabinding to the nucleotides of PSD-95/SAP90 mRNA. Combined with thebehavioral results above, it is suggested that the expression ofPSD-95/SAP90 in the spinal cord might be critical for spinal thermalhyperalgesia via NMDA receptor activation.

Example 3

[0039] This example demonstrates the expression and localization ofPSD95 in the spinal cord, as well as the colocalization with NMDAreceptors and nNOS.

[0040] To provide further support for the role of PSD-95/SAP90 in spinalhyperalgesia, we examined the expression of PSD-95/SAP90 and theinteraction of PSD-95/SAP90 with the NMDA receptor in the spinal cord.The regional expression and function of PSD-95/SAP90 in the mammalianbrain have been investigated using a variety of experimentalapproaches.^(3-5,7,9,16) To our knowledge, however, there are noprevious reports of its expression or function in adult spinal cord.Thus, RNA for messages encoding the PSD-95/SAP90 protein was extractedfrom tissues of the spinal cord, other regions of the brain (as positivecontrols), and muscle (as a negative control). This RNA was probed withthe use of RT-PCR analysis. A 0.735 Kb mRNA was detected in the spinalcord and regions of brain (hippocampus, cortex, cerebellum andbrainstem), but not in muscle (FIG. 1B). The PCR product then wasdirectly cloned into the TA cloning vector and verified as PSD-95/SAP90by automatic DNA sequencing. Furthermore, the distribution ofPSD-95/SAP90 immunoreactivity in the spinal cord was observed. Asillustrated in FIG. 2A, PSD-95/SAP90 immunoreactivity was found in thespinal cord and distributed mainly in lamina I and outer lamina II.Under high magnification, many PSD-95/SAP90 immunoreactive puncta wereobserved (FIG. 2B). The superficial dorsal horn not only contains manyinterneurons and their processes but also receives the processes fromthe deep dorsal horn neurons, the primary afferent termini from theperiphery and the descending fibers from supraspinal structures.²² SincePSD-95/SAP90 is specifically localized at synapses and has been foundboth pre- and post-synaptically in the brain,^(3,4) we investigated thesources of PSD-95/SAP90 immunoreactive puncta in the superficial dorsalhorn. In the dorsal root ganglion of normal rat, no PSD-95/SAP90 proteinwas detected (FIG. 1Aa). Also, there was no change in the density ofPSD-95/SAP90 immunoreactivity in the superficial dorsal horn afterunilateral spinal nerve cut or bilateral dorsolateral fasciculi cut(data not shown). More importantly, PSD-95/SAP90 mRNA was detected andPSD-95/SAP90 expression from antisense OND-treated rats wassignificantly suppressed in the spinal cord as described above. Thesedata indicate that PSD-95/SAP90 in the superficial dorsal horn, to agreat extent, is intrinsic to the spinal cord. The superficial dorsalhorn is the primary center for processing noxious stimulation.²² Thearea-specific expression and distribution of PSD-95/SAP90 in the spinalcord suggest that PSD-95/SAP90 has important implications for themechanisms of nociceptive processing at the spinal cord level. The NMDAreceptor has been demonstrated to mainly locate in lamina I and outerlamina II of the spinal cord.^(12,28) Combined with the present data,the NMDA receptor completely overlapped with PSD-95/SAP90 in the spinaldorsal horn. It is suggested that PSD-95/SAP90 may co-localize andinteract with the NMDA receptor in the spinal cord neurons. This wasfurther confirmed with the use of co-immunoprecipitation, demonstratingthat the PSD-95/SAP90 antibody was able to immunoprecipitate not onlyPSD-95/SAP90 but also NR2A/2B and nNOS in vivo (FIG. 3). In contrast,endothelial NOS (eNOS) was not immunoprecipitated with the PSD-95/SAP90antibody (FIG. 3). These findings show that PSD-95/SAP90 interacts withNR2A/2B in the spinal cord in vivo, suggesting that glutamatestimulation of the NMDA receptor in the spinal cord may result inassociation of the NMDA receptor with PSD-95/SAP90 protein in spinalhyperalgesia.

Example 4

[0041] This example describes the experimental procedures used in theexperiments described in the examples 1-3.

[0042] Animal preparation and behavioral testing. All experiments werecarried out with the approval of the Animal Care Committee at the JohnsHopkins University and were consistent with the ethical guidelines ofthe National Institutes of Health and the International Association forthe Study of Pain. Male Sprague-Dawley rats (250-300 g) were implantedwith an intrathecal PE-10 catheter into the subarachnoid space at therostral level of the spinal cord lumbar enlargement through an incisionat the atlanto-occipital membrane according to the method asdescribed.^(25,27) One week or more later, the rats were injectedintrathecally with saline or ONDs every 24 h for 4 days. On the fifthday, saline, NMDA, MK−801+NMDA or DNQX+NMDA was given intrathecally.Nociception was evaluated by the radiant heat tail-flick test. The dosesand time point of maximal effect of NMDA used in the present study weredetermined based on a previous study.²⁴ The tail-flick apparatus (Model33B Tail Flick Analgesy Meter, IITC Life Science, Woodland Hills,Calif., USA) generated a beam of radiant heat that was focused on theunderside of the tail, 5 cm from the tip. A cut-off time latency of 13.5s was used to avoid tissue damage to the tail. Nociception was assessedby the time required to induce tail-flick after applying radiant heat tothe skin of the tail. The latency of reflexive removal of the tail fromthe heat was measured automatically to the nearest 0.01 s. Tail-flicklatency was measured five times, and the basal latency was defined asthe mean. Tail-flick data were expressed as percentage change calculatedby the formula: (trial latency-baseline latency)/(baselinelatency)×100%. Finally, PE-10 catheter position from each animal wasconfirmed when lumbar enlargement segments were removed for western blotanalysis. PCR analysis of PSD-95/SAP90 in rat spinal cord. The cDNAsequences encoding portions of the PSD-95/SAP90 were amplified using thefollowing synthetic OND primers: PSD1 (5′-CAAGCCCAGCAATGCCTA-3′; SEQ IDNO: 3) and PSD2 (5′-CTTGTCGTAATCAAACAG-3′; SEQ ID NO: 4) foramplification of PSD-95/SAP90 codon positions 789-1525. RNA samples (1μg) from rat spinal cord, brain and muscle were reverse transcribed togenerate first-strand cDNA. The PCR reactions were performed for 25cycles. Each cycle included 30 s at 94° C., 30 s at 55° C., and 30 s at71° C. The PCR products were directly cloned into the TA cloning vector(Invitrogen Co., San Diego, CA, USA) and verified by automatic DNAsequencing. Fusion protein construction and preparation. cDNA sequenceencoding portion of PSD-95/SAP90 was amplified by PCR and subclonedin-frame into PGEX-2T (GIBCO, Rockville, Md., USA) via the BamHI andEcoRI restriction digest sites. The construct was then transformed intoBL21 bacteria, and following an induction of expression withisopropyl-β-D-thiogalactopyranoside, the protein was purified underdenaturing conditions using glutathione-coupled agarose. The aboveprotein was analyzed by SDS-PAGE followed by coomassie blue staining.Isolation of PSD fraction. PSD fraction was prepared according toprocedures described by Luo et al¹¹ with modifications. In brief, thespinal cord and brain from male Sprague-Dawley rats were homogenized andcentrifuged at 800×g for 10 min to recover the supernatant S1 and thepellet P1. The S1 fraction was subjected to centrifugation at 7,100×gfor 15 min to obtain the pellet P2 and the supernatant S2. P2 wasresuspended and again subjected to centrifugation at 8,200×g for 15 minto recover the synaptosomal fraction P2′. The P2′ fraction was treatedwith an osmotic shock by diluting with double-distilled water andfurther centrifuged at 25,000×g for 20 min to generate the pellet LP1and the supernatant LS1. LP1 was resuspended and centrifuged at 33,000×gfor 20 min. The pellet LP1P was resuspended and loaded onto adiscontinuous sucrose gradient composed of 0.10, 1.5 and 2.0 M sucrose.After ultracentrifugation at 208,000×g for 2 h, the PSD fraction wasrecovered at the interface between 0.5 and 2.0 M sucrose. The PSDfraction was finally resuspended and centrifuged at 208,000×g for 30min. The recovered the pellet, resuspended in buffer, was considered asthe purified PSD fraction. Co-immunoprecipitation and immunoblotting.About 2-4 μg of the affinity-purified mouse PSD-95/SAP90 antibody(Upstate Biotechnology, Lake Placid, N.Y., USA) was preincubated with100 μl of a 1:1 slurry of protein A-sepharose for 1 h, and theprotein-antibody complex was spun down at 2,000 rpm for 4 min. Thesolubilized PSD fraction (400 μg) was then added to the sepharose beadsand the mixture incubated for 2-3 h at 4° C. The mixture was washed oncewith 1% TritonX-100 in immunoprecipitation buffer (137 mM NaCl, 2.7 mMKCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 5 mM EGTA, 1 mM sodium vanadate, 10mM sodium pyrophosphate, 50 mM NaF, 20 U/ml Trasylol, and 0.1 mMphenylmethylsulfonyl fluoride), twice with 1% TritonX-100 inimmunoprecipitation buffer plus 300 mM NaCl, and three times withimmunoprecipitation buffer. The proteins were separated by SDS-PAGE andtransferred to a polyvinylidene difluoride membrane. In the controlgroups, PSD-95/SAP90 antibody was substituted with normal mouse serum,or was preincubated with excess of PSD-95/SAP90 fusion protein (100μg/ml). Immunoblotting was carried out as described by Lau et al ¹⁰.Individual proteins were detected with the use of primary antibodies toPSD-95/SAP90 (1:1000), NMDA receptor subunits 2A/2B (1:200, ChemiconInternational Inc, Temecula, Calif., USA), nNOS (1:2000, Santa CruzBiotechnology Inc., Santa Cruz, Calif., USA), eNOS (1:500, TransductionLab., Lexington, Ky., USA) and SAP102 (gift from Dr. R. L. Huganir).Immunocytochemistry. Rats were perfused with 4% paraformaldehyde in 0.1Mphosphate-buffered saline (PBS). The spinal cord was harvested andpostfixed at 4° C. for 4 h, and cryoprotected in 30% sucrose overnight.Sections (30 μm) were cut on a cryostat and then blocked for 1 h in PBScontaining 10% goat serum and 0.3% TritonX-100. Primary antibody toPSD-95/SAP90 (1: 1000) was diluted into blocking reagent and incubatedwith sections overnight. Immunoperoxidase histochemistry was performedusing the ABC method. Control sections lacking primary antiserum werestained in parallel.

Example 5

[0043] This example demonstrates the decrease in threshold forisoflurane caused by inhibition of expression of PSD 95.

[0044] The value for isoflurane MAC in the control (saline-treated)group was 1.16±0.08, which is consistent with that in the previousstudies.^(49,52) In the groups treated with the antisense ODNs at thedoses of 12.5, 25 and 50 μg, the isoflurane MACs were decreased fromisoflurane control MAC of 1%, 18% (P<0.01) and 44% (P<0.01),respectively (Table 1). In contrast, intrathecal administration of senseODN at the dose of 50 μg or missense ODN at the dose of 50 μg did notsignificantly change the value for isoflurane MAC compared to thecontrol group (Table 1).

[0045] No untoward effects were observed in any of the treated animalsincluding the antisense groups. In the ODN-treated groups, there was nosignificant change in either blood pressure or heart rate compared tocontrol group before the tail clamp (Table 2). Control baseline bloodpressure was 119.86±10.58 mmHg systolic and 106.36±7.78 mmHg diastolic,and control baseline heart rate was 513.00 40.28 beats/min.

Example 6

[0046] This example demonstrates that antisense ODN reduces thethreshold for isolfurane, even in the presence of NMDA which increasesthe threshold for isoflurane.

[0047] In the saline-treated group, intrathecal NMDA at a dose of 1.25μg caused an increase from isoflurane control MAC by 15% (P<0.01; FIG.1). The NMDA-induced change in isoflurane MAC was accompanied by asignificant increase in systolic and diastolic blood pressures(135.70±3.38 mmHg and 118.30±7.81 mmHg, respectively. P<0.05 vs control)but not in heart rate (529.20±5.20 beats/min, P>0.05 vs control).However, in the group pretreated with 50 μg of antisense ODN,intrathecal administration of 1.25 μg of NMDA did not result in asignificant increase in isoflurane MAC compared to the group treatedwith 50 μg of antisense ODN alone (P>0.05, FIG. 1). Interestingly, inthe group pretreated with 50 μg antisense ODN, intrathecal NMDA at adose of 1.25 μg still produced a significant increase in systolic anddiastolic blood pressures (138.00±_(5.77) mmHg and 117.00±6.35 mmHg,respectively. P<0.05 vs 50 μg antisense ODN-treated group alone) but notin heart rate (553±17 beats Imin, P>0.05 vs control).

Example 7

[0048] This example demonstrates that antisense ODN did not affectlocomotor function.

[0049] As shown in Table 3, ODNs with or without NMDA at the doses usedin the present study did not produce significant effects on locomotorfunction. Convulsions and hypermobility were not observed in any of thetreated animals including antisense ODN groups. In addition, there wasno significant difference in general behaviors including spontanousactivity between the control and the ODN-treated groups.

Example 8

[0050] This example demonstrates the materials and methods used inexamples 5-7.

[0051] The present study protocol was approved by the Animal CareCommittee at the Johns Hopkins University. Male Sprague-Dawley rats(250-300 g) were housed individually in cages on a standard 12 h -12 hlight-dark cycle. Water and food were available ad libitum until ratswere transported to the laboratory approximately 1 h before theexperiments. All experiments were performed under the same conditions.

[0052] Animal Preparation

[0053] Rats were anesthetized by intraperitoneal injection ofpentobarbital sodium (45 mg/kg). Chronic intrathecal catheters wereinserted by passing a polyethylene-10 (PE-10) catheter through anincision in the atlanto-occipital membrane to a position 8 cm caudal tothe cistema at the level of the lumbar subarachinoid space using themethods described previously.²⁷ The animals were allowed to recover for5-7 days before experiments were initiated. Rats that showedneurological deficits postoperatively were removed from the study.

[0054] To examine whether the deficiency of the expression ofPSD-95/SAP90 affected the threshold for isoflurane anesthesia, we madean antisense oligodeoxyribonucleotide (ODN) corresponding to thePSD-95/DLG/Z0-1 (PDZ) domain nucleotides 241 to 258(5′-TGTGATCTCCTCATACTC-3′; SEQ ID NO: 1) of rat PSD95/SAP90 mRNA, aswell as the sense ODN and missense ODN (5′-AAGCCCTTGTTCCCATTT-3′; SEQ IDNO: 2).⁴⁹ All of the ODNs were searched to exclude non-specificity ofthe sense or antisense ODNs and to show that missense ODN did not matchany confounding sequences in the GenBank database (GenBank accessionnumber M96853). The ODNs were dissolved in saline before administration.As described in the previous work,⁴⁹ the rats were injectedintrathecally with saline (10 μl) (control), antisense ODNs (12.5, 25,50 μg /10 μl ), sense ODN (50 μg /10 μl) and missense ODN (50 μg /10μl), respectively, followed by an injection of 10 μl of saline to flushthe catheter, every 24 h for 4 days.

[0055] Measurement of MAC

[0056] On the fifth day after saline or ODNs injection, each rat wasplaced in a clear plastic cone and anesthetized with 5% isoflurane inoxygen for three to five minutes. After tracheostomy, the trachea ofeach animal was intubated with a 16-gauge polyethylene catheter. Theinspired isoflurane concentration was reduced to 2%, and the animalsbreathed spontaneously until cannulation of a carotid artery and ajugular vein with PE-50 tubing was accomplished. The isofluraneconcentration was decreased further to 1.5%, and ventilation wascontrolled by a Harvard Animal Respirator (Harvard Apparatus, SouthNatick, Mass.) adjusted according to the measurement of arterial bloodgases to maintain normal partial pressure of oxygen (P_(o2)=91−94 mmHg),partial pressure of carbon dioxide (P_(CO2)33−41 mmHg) and pH(7.4-7.44). Electrocardiography and systolic and diastolic bloodpressure were monitored using a Grass Polygraph (Astroumed Grass,Quincy, Mass.) and Gould Pressure Transducer (Gould, Cleveland, Ohio).Rectal temperature was maintained between 36.5 and 37.5° C. by use of aheating blanket and warming lights.

[0057] A PE-10 catheter was introduced through and beyond theendotracheal tube until obstruction to passage was met and thenwithdrawn 1 to 2 mm. For isoflurane MAC measurement, the PE-10 catheterwas connected to a parameter airway gas monitor (Datex-Engstrom, Inc.,Tewksbury, Mass.). After stabilizing about 30 minutes, MAC was measuredaccording to the methods described previously³⁴ using a long hemostat(8-inch Rochester Dean Hemostatic Forceps) clamped to the first ratchetlock on the tail for 1 min. The tail was always stimulated proximal to aprevious test site. Gross movement of the head, extremities, or body wastaken as a positive test result, whereas grimacing, swallowing, chewing,or tail flick were considered negative results. The isofluraneconcentration was reduced in decrements of 0.12 to 0.15% until thenegative response became positive, with 12-15 min equilibration allowedafter changes in concentration.^(50,51) The MAC was considered to be theconcentration midway between the highest concentration that permittedmovement in response to the stimulus and the lowest concentration thatprevented movement. Finally, intrathecal PE-10 catheter position fromeach animal was confirmed.

[0058] In some saline-treated rats, after initial baseline MACdetermination, NMDA at the dose of 1.25 μg ³⁸ or saline was injectedintrathecally in a volume of 10 μl saline, followed by an injection of10 μl saline to flush the catheter. Fresh NMDA solution was prepared foreach experiment. An isoflurane concentration was chosen at whichmovement did not occur in the last negative response before the positivetest response. At this isoflurane concentration, 10 min after theintrathecal injection of NMDA, the animals were tested again forreactivity to tail clamp. The concentration of isoflurane was increased,and response to tail clamp was checked every 12-15 min thereafter untila negative response was achieved. In some antisense ODN (50 μg)-treatedrats, after initial MAC determination, NMDA or saline was alsoadministered intrathecally. The MAC for isoflurane was again determinedfollowing the aforementioned procedures.

[0059] Tests of Locomotor Function

[0060] The effects of ODNs on locomotor function were examined using thefollowing methods.⁵² The animals were organized randomly into sixgroups: control (saline); 12.5 μg antisense ODN; 25 μg antisense ODN; 50μg antisense ODN; 50 μg sense ODN; 50 μg missense ODN. The rats werepretreated with ODNs or saline in the manner described above. On thefifth day, 10 μl of saline was injected intrathecally for each rat. Insome saline or antisense ODN (50 μg)-treated rats, fresh NMDA solution(1.25 μg /10 μl) was injected intrathecally. The following tests wereperformed with the experimenter blind to which group was treated withthe agents: (1) Placing reflex: The rat was held with the hind limbsslightly lower than the forelimbs, and the dorsal surfaces of the hindpaws were brought into contact with the edge of a table. Theexperimenter recorded whether the hind paws were placed on the tablesurface reflexively; (2) Grasping reflex: The rat was placed on a wiregrid and the experimenter recorded whether the hind paws grasped thewire on contact; (3) Righting reflex: The rat was placed on its back ona flat surface and the experimenter noted whether it immediately assumedthe normal upright position. Scores for placing, grasping and rightingreflexes were based on counts of each normal reflex exhibited in fivetrials. In addition, the rat general behaviors including spontaneousactivity were observed.

[0061] Statistical Analysis

[0062] The MAC data were assessed statistically by an analysis ofvariance. Intergroup differences were analyzed using the Newman-Keulstest. Locomotor data were assessed by a rank sum test. All data arereported as the mean ±SD. Significance was set at P<0.05.

Example 9

[0063] This example demonstrates the role of PSD95 in formalin-inducedpain, which is a model for inflammatory-induced pain.

[0064] Pretreatment with PSD-95 antisense ODN produced significantdecreases in formalin-induced pain behaviors and c-fos expression in thespinal cord. Intrathecal antisense ODN at 50 μg reduced the number offlinches and shakes evoked by formalin by 59% (p<0.01) in the tonicperiod but not in the phasic period. At the same dose, the antisense ODNalso decreased the number of Fos-like immunoreactive neurons per sectionby 48% (p<0.05). However, the antisense ODN at 12.5 and 25 μg failed toproduce significant changes in the number of flinches and shakes in thephasic and tonic periods, or in the number of Fos-like immunoreactiveneurons, when compared to the saline-treated group. Similarly, the senseODN- and the missense ODN-treated groups did not show any significantdifference in the number of flinches and shakes in either period, whencompared to the saline-treated group.

[0065] These results demonstrate that PSD-95 antisense significantlyreduced formalin-induced nociceptive behaviors in the tonic period butnot in the phasic period. This suggests that PSD-95 protein may play akey role in the spinal sensitization induced by subcutaneous formalininjection.

[0066] All of the experiments were carried out with the approval of theAnimal Care Committee at the Johns Hopkins University. Thirty-eight maleSprague-Dawley rats (250-300 g, Hilltop Laboratory Animals, Scottsdale,Pa., USA) were implanted with an intrathecal PE-10 catheter at therostral level of the spinal cord lumbar enlargement according to themethod described by Yaksh and Rudy. After 4 to 7 days of recovery, theywere intrathecally injected with one of the following agents every 24hours for 4 consecutive days: saline (10 μl, n−6), PSD-95 antisenseoligodeoxynucleotide (ODN) (12.5 μg/10 μl, n−6; 25 μg/10 μl, n=6; 50μg/10 μl, n=6), sense ODN (50 μg/10 μl, n=5) or missense ODN (50 μg/10μl, n=9). On the fifth day, formalin (4%, 100 μl) was injected into oneof the hindpaws. The number of flinches and shakes of the injected pawwas assessed for 1 hour. The observational session was divided intophasic (0-10min) and tonic (10-60min) periods. Rats were sacrificed twohours after formalin injection and their lumbar spinal cords wereharvested for c-fos immunohistochemistry.

[0067] Data were assessed as mean ±SD. Behavioral test andimmunohistochemistry results were assessed by ANOVA. Post-hoc testingwas conducted using Bonferroni test. Significance was set at p<0.05.

Example 10

[0068] This example demonstrates that halothane inhibits the NMDAreceptor signaling pathway by inhibiting PDZ domain interactions betweenPSD-95 or PSD-93 and NMDA receptors or nNOS.

[0069] Under normal conditions, PSD-95 interacts with nNOS, resulting ingood growth of the yeast carrying pGAD424-PSD-95 and pGBT9-nNOS in -LTHmedium. We found that halothane dose-dependently inhibited the growth ofthe yeast in -LTH media. Treatment with low halothane concentrations(0.4%-0.7%) slowed the growth of yeast clones. At high concentration(1.3%), halothane completely inhibited yeast growth. A similarphenomenon was observed in the growth of the yeast carryingpGAD424-PSD-95 and pGBT9-2B. The growth of the yeast carryingpGAD424-PSD-93 and pGBT9-nNOS or 2B was also inhibited by halothane in asimilar way. However, when these yeast clones grew in -LT instead of-LTH media in the presence of high halothane concentration (3.6%),growth did not differ from yeast grown without halothane. This findingsuggests that halothane is not cytotoxic to yeast. Rather, the failureof yeast to grow in -LTH media in the presence of halothane must be dueto disruption of protein-protein interactions by halothane. In addition,we used a biochemistry approach to demonstrate that halothane blocksGST-fusion PSD-95 or PSD-93 protein from binding to rat brain NMDAreceptors or to nNOS. These findings confirm the yeast two-hybridresults.

[0070] We utilized the yeast two-hybrid system to investigate theeffects of halothane on protein interactions within the NMDA receptorsignaling complex. The PDZ domain of nNOS or the C-terminus of NMDAreceptor subunit 2B (NR2B) was fused in frame with the GAL4 DNA-bindingdomain in a yeast vector, pGBT9. The PDZ domain of PSD95 or PSD-93 wasfused in frame with the GAL4 activation domain in another yeast vector,pGAD424. Both yeast vectors were co-transformed into the Y190 yeaststrain, which was then grown in the absence or presence of halothane atclinically relevant concentrations. Protein-protein interactions wereconfirmed by both yeast growth on -Leu/-Trp/-His (-LTH) medium and laczexpression. To confirm the yeast two-hybrid results, the GST fusionprotein binding assay was performed. The GST-fusion proteins, consistingof the second PDZ domain of PSD-95 or PSD-93, were expressed inbacterial BL21 cells and purified using glutathione-coupled agarose.After preincubation with or without different concentrations ofhalothane, GST-PSD-95 or GST-PSD-93 was incubated with membrane proteinsfrom rat hippocampus at room temperature for 1 h. After extensivewashing, the bound proteins were eluted by boiling in 1 X SDS-PAGEsample buffer and detected by immunoblotting.

[0071] Utilizing both the yeast two-hybrid system and protein bindingassays, we found that halothane dose-dependently inhibited proteininteractions of PSD-95/NMDA receptor, PSD-95/nNOS, PSD-93/NMDA receptorand PSD-93/nNOS at physiological concentration. These proteininterconnections within the NMDA receptor signaling complex are believedto be critical for excitatory synaptic signal transduction. Disruptionof the signal complex may shed light on a novel mechanism for generalanesthesia.

Example 11

[0072] This example demonstrates the interaction of PSD-95/SAP90 withNMDA receptor and neuronal nitric oxide synthase (nNOS) were examined.

[0073] We probed RNA from tissures of the spinal cord, other regions ofbrain (as positive control) and muscle (as negative control) formessages encoding the PSD-95/SAP90 protein with the use of RT-PCRanalysis. A 0.735 Kb mRNA was detected in the spinal cord and theregions of brain but not in muscle. The PCR product was directly clonedinto the TA cloning vector and verified as PSD-95/SAP90 by automatic DNAsequencing. PSD-95/SAP90 protein also was found to enrich in thepostsynaptic density fraction of the spinal cord. Moreover,immunohistochemistry showed that PSD-95/SAP90 was distributed mainly inspinal superficial laminae, where PSD-95/SAP90 overlapped with NMDAreceptor subunits 2A/2B (NR2A/2B) and nNOS, suggesting that PSD-95/SAP90might interact with NR2A/2B and nNOS in the spinal cord. This wasconfirmed with the use of co-immunoprecipitation, demonstrating that thePSD-95/SAP90 antibody was able to immunoprecipitate not onlyPSD-95/SAP90 but also NR2A/2B and nNOS in vivo. In contrast, endothelialNOS was not immunoprecipitated with PSD-95/SAP90 antibody. Thearea-specific expression of PSD-95/SAP90 and its interaction with NMDAreceptor and nNOS in the spinal cord in the present study suggestPSD-95/SAP90 may have important implications for the mechanisms ofnociceptive processing.

Example 12

[0074] This examples demonstrates the role of PSD-95/SAP90 in chronicneuropathic pain.

[0075] The effect of the deficiency of PSD-95/SAP90 on mechanical andthermal hyperalgesia in a rat neuropathic pain model was observed. Theantisense oligonucleotide (OND) specifically against PSD-95/SAP90 wasemployed to reduce the expression of PSD-95/SAP90 in spinal cord. Therats were injected intrathecally with saline (10 μl), antisense OND (50μg/10 μl) or sense OND (50 μg/10 μl) every 24 h for 4 days. Theunilateral L5 spinal nerve was ligated. Hind paw withdrawal response tomechanical or heat stimuli was conducted 1 day prior to the surgery andat 3, 5, 7 and 9 days postoperatively. In the saline-treated group,mechanical and thermal hyperalgesia developed within 3 days andpersisted for 9 days or longer. The pretreatment of antisense but notsense ODN resulted in a significant delay of the onset of the mechanicaland thermal hyperalgesia. Our results indicate that the deficiency ofPSD-95/SAP90 delayed the development of the neuropathic pain.PSD-95/SAP90 is likely involved in the molecular mechanism of theproduction of hyperalgesia in neuropathic pain triggered via NMDAreceptor activation.

REFERENCES

[0076] 1. Aanonsen L. M., Lei S., and Wilcox G. L. (1990) Excitatoryamino acid receptors and nociceptive neurotransmission in rat spinalcord. Pain 41, 309-321.

[0077] 2. Brenman J. E., Christopherson K. S., Craven S. E., McGee A. W.and Bredt D. S. (1996) Cloning and characterization of postsynapticdensity 93, a nitric oxide synthase interacting protein. J. Neurosci.16, 7407-7415.

[0078] 3. Brenman J. E., Chao D. S., Gee S. H., McGee A. W., Craven S.E., Santillano D. R., Wu Z., Huang F., Xia H., Peters M. F., Froehner S.C. and Bredt D. S. (1996) Interaction of nitric oxide synthase with thepostsynaptic density protein PSD-95 and α1-syntrophin mediated by PDZdomains. Cell 84, 757-767.

[0079] 4. Cho K. O., Hunt C. A. and Kennedy M. B. (1992) The rat brainpostsynaptic density fraction contains a homology of the drosophiladiscs-large tumor suppressor protein. Neuron 9, 929-942.

[0080] 5. Christopherson K. S., Hillier B. J., Lim W. A. and Bredt D. S.(1999) PSD-95 assembles a ternary complex with the N-methyl-D-asparticacid receptor and a bivalent neuronal NO synthase PDZ domain. J. Biol.Chem. 274, 27467-27473.

[0081] 6. Kim E., Cho K. O., Rothschild A. and Sheng M. (1996)Heteromultimerization and NMDA receptor-clustering activity ofchapsyn-110, a member of the PSD-95 family of proteins. Neuron 17,103-113.

[0082] 7. Kistner U., Wenzel B. M., Veh R. W., Cases-Langhoff C., GarnerA. M., Appeltauer U., Voss B., Gundelfinger E. D. and Garner C. C.(1993) SAP90, a rat presynaptic protein related to the product of thedrosophila tumor suppressor gene dig-A. J. Biol. Chem. 268,4580-4583.

[0083] 8. Kolhekar R., Meller S. T. and Gebhart G. F. (1993)Characterization of the role of spinal N-methyl-D-aspartate receptors inthermal nociception in the rat. Neuroscience 57, 385-395.

[0084] 9. Kornau H. C., Schenker L. T., Kennedy M. B. and Seeburg P. H.(1995) Domain interaction between MDA receptor subunits and thepostsynaptic density protein PSD-95. Science 269, 1737-1740.

[0085] 10. Lau L.-H., Mammen A., Ehlers M. D., Kindler S., Chung W. J.,Garner C. C. and Huganir R. L. (1996) Interaction of theN-methyl-D-aspartate receptor complex with a novel synapse-associatedprotein, SAP-102. J. Biol. Chem. 271, 21622-21628.

[0086] 11. Luo J., Wang Y., Yasuda R. P., Dunah A. W. and Wolfe B. W.(1997) The majority of N-mehtyl-D-aspartate receptor complexes in adultrat cerebral cortex contain at least three different subunits. Mol.Pharmacol. 51, 79-86.

[0087] 12. Marvizon J. C., Martinez V., Grady E. F., Bunnett N. W. andMayer E. A. (1997) Neurokinin 1 receptor internalization in spinal cordslices induced by dorsal root stimulation is mediated by NMDA receptors.J. Neurosci. 17, 8129-36

[0088] 13. Meller S. T., Dykstra C. and Gebhart G. F. (1992) Productionof endogenous nitric oxide and activation of soluble guanylate cyclaseare required for N-methyl-D-aspartate-produced facilitation of thenociceptive tail-flick reflex. Eur. J. Pharmacol. 214,93-96.

[0089] 14. Meller S. T., Dykstra C. and Gebhart G. F. (1996) Acutethermal hyperalgesia in the rat is produced by activation ofN-methyl-D-aspartate receptors and protein kinase C and production ofnitric oxide. Neuroscience 71, 327-335.

[0090] 15. Meller S. T. and Gebhart G. F. (1993) Nitric oxide (NO) andnociceptive processing in the spinal cord. Pain 52, 127-136.

[0091] 16. Migaud M., Charlesworth P., Dempster M., Webster L. C.,Watabe A. W., Makhinson M., He Y., Ramsay M. F., Morris R. G., MorrisonJ. H., O'Dell T. J. and Grant S. G. (1998) Enhanced long-termpotentiation and impaired learning in mice with mutant postsynapticdensity-95 protein. Nature 396, 433-439.

[0092] 17. Muller B. M., Kistner U., Kindler S., Chung W. J., KuhlendahlS., Fenster S. D., Lau L. F., Veh R. W., Huganir R. L., Gundelfinger E.D. and Garner C. C. (1996) SAP102, a novel postsynaptic protein thatinteracts with NMDA receptor complexes in vivo. Neuron 17, 255-265.

[0093] 18. Myers K. J. and Dean N. M. (2000) Sensible use of antisense:how to use oligonucleotides as research tools. TiPS 21, 19-23.

[0094] 19. Nakanishi S. (1992) Molecular diversity of glutamatereceptors and implications for brain function. Science 258, 597-603.

[0095] 20. Niethammer M., Kim E. and Sheng M. (1996) Interaction betweenthe C terminus of NMDA receptor subunits and multiple membranes of thePSD-95 family of membrane-associated guanylate kinase. J. Neurosci. 16,2157-2163.

[0096] 21. Randic M., Jiang M. C. and Cerne R. (1993) Long-termpotentiation and long-term depression of primary afferentneurotransmission in the spinal cord. J. Neurosci. 13, 5228-5241.

[0097] 22. Rustioni A. and Weinberg R. J. (1989) The somatosensorysystem. In: Handbook of Chemical Neuroanatomy (Bjorklund A, Hokfelt T,Swanson LW, eds), pp219-321. Amsterdam: Elsevier.

[0098] 23. Sattler R., Xiong Z., Lu W.-Y., Hafner M., MacDonald J. F.and Tymianski M. (1999) Specific coupling of NMDA receptor activation tonitric oxide neurotoxicity by PSD-95 protein. Science 284, 1845-1848.

[0099] 24. Siegan J. B. and Sagen J. (1995) Attenuation of NMDA-inducedspinal hypersensitivity by adrenal medullary transplants. Brain Res.680, 88-98.

[0100] 25. Tao Y.-X., Hassan A., Haddad E. and Johns R. A. (2000)Expression and action of cyclic GMP-dependent protein kinase Iα ininflammatory hyperalgesia in rat spinal cord. Neuroscience 95, 525-533.

[0101] 26. Woolf C. J. and Thompson S. W. N. (1991) The induction andmaintenance of central sensitization is dependent on N-methyl-D-asparticacid receptor activation:

[0102] implications for the treatment of post-injury painhypersensitivity states. Pain 44, 293-299.

[0103] 27. Yaksh T. L. and Rudy T. A. (1976) Analgesia mediated by adirect spinal action of narcotics. Science 192, 1357-1358.

[0104] 28. Yung K. K. (1998) Localization of glutamate receptors indorsal horn of rat spinal cord. Neuroreport 9, 1639-1644

[0105] 29. Aanonsen LM, Wilcox GL: Nociceptive action of excitatoryamino acids in the mouse: effects of spinally administered opioids,phencyclidine and sigma agonists. J Pharmacol Exp Ther 1987; 243: 9-19.

[0106] 30. Dickenson AH, Aydar E: Antagonism at the glycine site on theNMDA receptor reduces spinal nociception in the rat. Neurosci Lett 1991;121: 263-266.

[0107] 31. Kuroda Y, Strebel S, Rafferty C, Bullock R: Neuroprotectivedoses of N-Methyl-D-aspartate receptor antagonists profoundly reduce theminimum alveolar anesthetic concentration (MAC) for isoflurane in rats.Anesth Analg 1993; 77: 795-800.

[0108] 32. Ishizaki K, Yoon DM, Yoshida N, Yamazaki M, Arai K, Fujita T:Intrathecal administration of N-Methyl-D-aspartate receptor antagonistreduces the minimum alveolar anesthetic concentration of isoflurane inrats. Br J Anaesth 1995; 75: 636-638.

[0109] 33. Ishizaki K, Yoshida N, Yoon DM, Yoon MH, Sudoh M, Fujita T:Intrathecally administered NMDA receptor antagonists reduce the MAC ofisoflurane in rats. Can J Anaesth 1996; 43: 724-730.

[0110] 34. Davies SN, Lodge D: Evidence for involvement ofN-methyl-D-aspartate receptors in ‘wind-up’ of class 2 neurons in thedorsal horn of the rat. Brain Res 1987; 424: 402-406.

[0111] 35. Dickenson AH, Sullivan AF: Evidence for a role of the NMDAreceptor in the frequency dependent potentiation of deep dorsal hornneurons following C-fiber stimulation. Neuropharnacology 1987; 26:1235-1238.

[0112] 36. Dougherty PM, Willis WD: Enhancement of spinalthalamic neuronresponses to chemical and mechanical stimuli following combinedmicro-iontophoretic application of N-methyl-D-aspartic acid andsubstance P. Pain 1991; 47: 85-93.

[0113] 37. Malmberg AB, Yaksh TL: Hyperalgesia mediated by spinalglutamate or substance P receptor blocked by spinal cyclooxygenaseinhibition. Science 1992;

[0114]257: 1276-1279.

[0115] 38. Kawamata T, Omote K: Activation of spinalN-methyl-D-aspartate receptors stimulates a nitric oxide/cyclicguanosine 3,5-monophosphate/glutamate release cascade in nociceptivesignaling. Anesthesiology 1999; 91: 1415-1424.

[0116] 39. Tao YX, Johns RA: Activation of cGMP-dependent protein kinaseIα is required for N-methyl-D-aspartate- or nitric oxide-produced spinalthermal hyperalgesia. Eur J Pharmacol 2000; 392: 141-145.

[0117] 40. Davar G, Hama A, Deykin A, Vos B, Maciewicz R: MK-801 blocksthe development of thermal hyperalgesia in a rat model of experimentalpainful neuropathy. Brain Res 1991; 553: 327-330.

[0118] 41. Mao J, Price DD, Mayer DJ, Lu J, Hayes RL: Intrathecal MK-801and local nerve anesthesia synergistically reduce nociceptive behaviorsin rats with peripherial mononeuropathy. Brain Res 1992; 576: 254-262.

[0119] 42. Ren K, Dubner R: NMDA receptor antagonists attenuatemechanical hyperalgesia in rats with unilateral inflammation of thehindpaw. Neurosci Lett 1993; 163: 22-26.

[0120] 43. Seltzer Z, Cohn S, Ginzburg R, Behavior in rats by spinaldisinhibition and NMDA receptor blockade of injury discharge. Pain 1991;45: 69-75.

[0121] 44. Yamamoto T, Shimoyama N, Mizuguchi T: The effect of morphine,MK-801, an NMDA antagonist, and CP-96,345, an NK-1 antagonist, on thehyperalgesia evoked by carrageenan injection in the rat paw.Anesthesiology 1993; 78: 124-133.

[0122] 45. Kennedy MB: The postsynaptic density at glutamatergicsynapses. Trends Neurosci 1997; 20: 264-268.

[0123] 46. Nagano T, Jourdi H, Nawa H: Emerging roles of Dlg-like PDZprotein in the organization of the NMDA-type glutamatergic synapse. JBiochem 1998; 124: 869-875.

[0124] 47. Hata Y, Nakanishi H, Takai Y: Synaptic PDZ domain-containingproteins. Neurosci Res 1998; 32: 1-7.

[0125] 48. O'Brien RJ, Lau LF, Huganir RL: Molecular mechanisms ofglutamate receptor clustering at excitatory synapses. Curr OpinNeurobiol 1998; 8: 364-369.

[0126] 49. Tao YX, Huang YZ, Mei L, Johns RA: Expression of PSD-95/SAP90is critical for N-methyl-D-aspartic acid receptor-mediated thermalhyperalgesia in the spinal cord. Neuroscience 2000; 98: 201-206.

[0127] 50. Tao YX, Hassan A, Johns RA: Intrathecally administeredcGMP-dependent protein kinase Iα inhibitor significantly reduced thethreshold for isoflurane anesthesia. Anesthesiology; 2000: 493-499.

[0128] 51. Eger EI, Saidman LJ, Brandstater B: Minimum alveolaranesthetic concentration:

[0129] A standard of anesthetic potency. Anesthesiology 1965; 26:756-763.

[0130] 52. Eger EI: Effect of inspired anesthetic concentration on therate of rise of alveolar concentration. Anesthesiology 1963; 26:153-157.

[0131] 53. Coderre TJ, Van Empel I: The utility of excitatory amino acid(EAA) antagonist as analgesic agents. I. Comparison of theantinociceptive activity of various classes of EAA antagonist inmechanical, thermal and chemical nociceptive tests. Pain 1994; 59:345-352.

[0132] 54. Pajewski TN, DiFazio CA, Moscicki JC, Johns RA: Nitric oxidesynthase inhibitor, 7-nitro indazole and nitroG-L-arginine methyl ester,dose dependently reduce the threshold for isoflurane anesthesia.Anesthesiology 1996; 85: 1111-1119.

[0133] 55. Hunt CA, Schenker LJ, Kennedy MB: PSD-95 is associated withthe postsynaptic density and not with the presynaptic membrane atforebrain synapses. J Neurosci 1996; 16: 1380-1388.

[0134] 56. Valtschanoff JG, Burette A, Wenthold RJ, Weinberg RJ:Expression of NR2 receptor subunit in rat somatic sensory cortex:synaptic distribution and colocalization with NR1 and PSD-95. J CompNeurol 1999; 410: 599-611.

[0135] 57. Garcia RA, Vasudevan K, Buonanno A: The neuregulin receptorErbB-4 interacts with PDZ-containing proteins at neuronal synapses. ProcNatl Acad Sci USA 2000; 97: 3596-601.

[0136] 58. Coote JH: The organization of cardiovascular neurons in thespinal cord. Rev Physiol Pharmacol 1988; 110: 147-285.

[0137] 59. Loewy AD, Spyer KM: Central Regulation of autonomicfunctions. Oxford, UK:

[0138] Oxford Uni Press, 1999.

[0139] 60. Morrison SF, Callaway TA, Milner TA, Reis DJ: Glutamate inthe spinal sympathetic intermediolateral nucleus: location by light andelectron microscopy. Brain Res 1989; 503: 5-15.

[0140] 61. Morrison SF, Emsberger P, Milner TA, Callaway TA, Gong A,Reis DJ: A glutamate mechanism in the intermediolateral nucleus mediatessympathoexcitatory responses to stimulation of the rostral ventrolateralmedulla. Prog Brain Res 1989; 81: 159-169.

[0141] 62. Hong Y, Henry JL: Glutamate, NMDA and NMDA receptorantagonists:

[0142] cardiovascular effects of intrathecal administration in the rat.Brain Res 1992; 569: 38-45.

[0143] 63. West M, Huang W: Spinal cord excitatory amino acids andcardiovascular autonomic responses. Am J Physiol 1994; 267: H865-873.

[0144] 64. Hong Y, Yashpal K, Henry JL: Cardiovascular responses tointrathecal administration of strychnine in the rat: Brain Res 1989;169-173. TABLE 1 Effects of the suppression of the expression ofPSD-95/SAP90 in the spinal cord on the N-methyl-o-aspartate-inducedthermal hyperalgesia MK-801 + DNQX + AS (25 μg) + AS (50 μg) + SE (50μg) + MS (50 μg) + Control NMDA NMDA NMDA NMDA NMDA NMDA NMDA ΔTF −1.23± 1.48 −25.84 ± 1.91* 0.9 ± 3.0*** −22.6 ± 3.13* −11.65 ± 2.46**^(,)****−4.72 ± 2.49*** −21.48 ± 1.55* −20.96 ± 1.68* latency (%)

[0145] TABLE 2 Effects of Antisense (AS), Sense (SE), and Missense (SE)Oligodeoxyribonucleotides and Saline on Isoflurane MAC, Blood Pressure(BP), and Heart Rate Saline 12.5 μg AS 25 μg AS 50 μg AS 50 μg SE 50 μgMS (n = 14) (n = 6) (n = 6) (n = 6) (n = 6) (n = 6) MAC  1.16 ± 0.08 1.15 ± 0.18  0.98 ± 0.14*  0.72 ± 0.05*  1.15 ± 0.21  1.13 ± 0.15 BP(mmHg) Systolic 119.86 ± 10.58 127.58 ± 11.72 122.75 ± 10.81 129.58 ±11.73 126.67 ± 10.40 121.33 ± 15.84 Diastolic 106.36 ± 7.78  112.58 ±7.14  105.83 ± 7.89  112.50 ± 11.20 105.58 ± 13.07 105.75 ± 11.40 Heartrate 513.00 ± 40.78 534.80 ± 29.13 541.20 ± 16.70 514.20 ± 62.20 529.60± 22.61 524.70 ± 44.90 (beats/min)

[0146] TABLE 3 Mean (SD) Changes In Locomotor Test Agents PlacingGrasping Righting Saline 5 (0) 5 (0) 5 (0) 12.5 μg AS 5 (0) 5 (0) 5 (0)25 μg AS 5 (0) 5 (0) 5 (0) 50 μg AS 4.83 (0.41) 4.67 (0.52) 4.83 (0.41)50 μg SE 5 (0) 5 (0) 5 (0) 50 μg MS 5 (0) 5 (0) 5 (0) Saline + 1.25 μg 5(0) 5 (0) 5 (0) NMDA 50 μg AS + 1.25 μg 4.83 (0.41) 4.83 (0.41) 4.83(0.41) NMDA

[0147]

1 4 1 18 DNA Rattus rattus 1 tgtgatctcc tcatactc 18 2 18 DNA Rattusrattus 2 aagcccttgt tcccattt 18 3 18 DNA Artificial Sequence PCR primer3 caagcccagc aatgccta 18 4 18 DNA Artificial Sequence PCR primer 4cttgtcgtaa tcaaacag 18

1. A method for relieving acute or chronic pain comprising:administering to a subject in need thereof an effective amount of anagent which inhibits expression of PSD93 or PSD95, whereby acute orchronic pain experienced by the subject is relieved.
 2. The method ofclaim 1 wherein the agent is an antisense oligonucleotide which iscomplementary to mRNA encoding PSD93.
 3. The method of claim 1 whereinthe agent is an antisense oligonucleotide which is complementary to mRNAencoding PSD95.
 4. The method of claim 1 wherein the antisenseoligonucleotide is complementary to nucleotides encoding a PDZ domain.5. The method of claim 3 wherein the antisense oligonucleotide iscomplementary to nucleotides 241 to
 258. 6. The method of claim 1wherein the agent is administered intrathecally.
 7. A method fortreating or preventing hyperalgesia comprising: administering to asubject in need thereof an effective amount of an agent which inhibitsexpression of PSD93 or PSD95, whereby hyperalgesia experienced by thesubject is relieved.
 8. The method of claim 7 wherein the agent is anantisense oligonucleotide which is complementary to mRNA encoding PSD93.9. The method of claim 7 wherein the agent is an antisenseoligonucleotide which is complementary to mRNA encoding PSD95.
 10. Themethod of claim 7 wherein the antisense oligonucleotide is complementaryto nucleotides encoding a PDZ domain.
 11. The method of claim 9 whereinthe antisense oligonucleotide is complementary to nucleotides 241 to258.
 12. The method of claim 7 wherein the agent is administeredintrathecally.
 13. A method of reducing a threshold for anesthesiacomprising: administering to a subject an anesthetic and an agent whichinhibits expression of PSD93 or PSD95, wherein the amount of anestheticadministered is less than the amount required in the absence of theagent to achieve a desired anesthetic effect, whereby the desiredanesthetic effect is achieved.
 14. The method of claim 13 wherein theagent is an antisense oligonucleotide which is complementary to mRNAencoding PSD93.
 15. The method of claim 13 wherein the agent is anantisense oligonucleotide which is complementary to MRNA encoding PSD95.16. The method of claim 13 wherein the antisense oligonucleotide iscomplementary to nucleotides encoding a PDZ domain.
 17. The method ofclaim 15 wherein the antisense oligonucleotide is complementary tonucleotides 241 to
 258. 18. The method of claim 13 wherein the agent isadministered intrathecally.
 19. A pharmaceutical formulation comprisingan isolated and purified antisense polynucleotide which is complementaryto PSD95 or PSD93 mRNA.
 20. The pharmaceutical formulation of claim 19wherein the polynucleotide is complementary to nucleotides encoding aPDZ domain.
 21. The pharmaceutical formulation of claim 19 wherein thepolynucleotide is complementary to nucleotides encoding a C-terminal PDZdomain.
 22. The pharmaceutical formulation of claim 19 wherein thepolynucleotide is complementary to nucleotides 241 to 258 of PSD95. 23.The pharmaceutical formulation of claim 19 wherein the polynucleotide iscomplementary to PSD93 MRNA.
 24. The pharmaceutical formulation of claim19 wherein the polynucleotide is manufactured under regulatory-approvedconditions for administration to humans.
 25. The pharmaceuticalformulation of claim 19 wherein the polynucleotide is pyrogen-free. 26.A method for relieving acute or chronic pain comprising: administeringto a subject in need thereof an effective amount of an agent whichinhibits interaction of a first protein selected from the groupconsisting of PSD93 and PSD95, with a second protein selected from thegroup consisting of nNOS and NMDA receptor, wherein the agent does notcause cardiovascular or respiratory depression, whereby acute or chronicpain experienced by the subject is relieved.
 27. The method of claim 26wherein the agent is administered intrathecally.
 28. A method fortreating or preventing hyperalgesia comprising: administering to asubject in need thereof an effective amount of an agent which inhibitsinteraction of a first protein selected from the group consisting ofPSD93 and PSD95, with a second protein selected from the groupconsisting of nNOS and NMDA receptor, wherein the agent does not causecardiovascular or respiratory depression, whereby hyperalgesiaexperienced by the subject is relieved.
 29. The method of claim 28wherein the agent is administered intrathecally.
 30. A method ofreducing a threshold for anesthesia comprising: administering to asubject an anesthetic and an agent which inhibits interaction of a firstprotein selected from the group consisting of PSD93 and PSD95, with asecond protein selected from the group consisting of nNOS and NMDAreceptor, wherein the agent does not cause cardiovascular or respiratorydepression, wherein the amount of anesthetic administered is less thanthe amount required in the absence of the agent to achieve a desiredanesthetic effect, whereby the desired anesthetic effect is achieved.31. The method of claim 30 wherein the agent is administeredintrathecally.
 32. The method of claim 26, 28, or 30 wherein the agentbinds to a PDZ domain of the first or second protein.
 33. The method ofclaim 26, 28, or 30 wherein the agent does not impair motor function.34. The method of claim 13 or 30 wherein the anesthetic is selected fromthe group consisting of halothane, isoflurane, desflurane, xenon, andsevoflurane.
 35. A method of anesthetizing a subject comprising:administering to a subject an agent which inhibits expression of PSD93or PSD95, whereby the agent renders the subject unconscious or sedated.36. The method of claim 35 wherein the agent is an antisenseoligonucleotide which is complementary to mRNA encoding PSD93.
 37. Themethod of claim 35 wherein the agent is an antisense oligonucleotidewhich is complementary to mRNA encoding PSD95.
 38. The method of claim35 wherein the antisense oligonucleotide is complementary to nucleotidesencoding a PDZ domain.
 39. The method of claim 37 wherein the antisenseoligonucleotide is complementary to nucleotides 241 to
 258. 40. Themethod of claim 35 wherein the agent is adminstered intrathecally.
 41. Amethod of anesthetizing a subject comprising: administering to a subjectan agent which inhibits interaction of a first protein selected from thegroup consisting of PSD93 and PSD95, with a second protein selected fromthe group consisting of nNOS and NMDA receptor, wherein the agent doesnot cause cardiovascular or respiratory depression, whereby the agentrenders the subject unconscious or sedated.
 42. The method of claim 41wherein the agent is administered intrathecally.
 43. The method of claim41 wherein the agent binds to a PDZ domain of the first or secondprotein.
 44. The method of claim 41 wherein the agent does not impairmotor function.
 45. A method of screening for substances useful forrelieving pain or inducing unconsciousness or sedation, comprising:contacting a test substance with a first protein and a second proteinunder conditions where the first protein and the second protein bind toeach other, wherein the first protein is selected from the groupconsisting of PSD93, PSD95, and a combination thereof, wherein thesecond protein is selected from the group consisting of nNOS, NMDAreceptor, NR2A subunit, NR2B subunit, and combinations thereof;determining an amount selected from the group consisting of: free nNOS,free PSD93, free PSD95, free NMDA receptor, free NR2A subunit, free NR2Bsubunit, bound nNOS, bound PSD93, bound PSD95, bound NMDA receptor,bound NR2A subunit, bound NR2B subunit and combinations thereof;identifying a test substance which increases the amount of free nNOS,free PSD93, free PSD95, free NMDA receptor, free NR2A subunit, or freeNR2B subunit, or which decreases the amount of bound nNOS, bound PSD93,bound PSD95, bound NMDA receptor, bound NR2A subunit, or bound NR2Bsubunit as a candidate drug for relieving pain or inducingunconsciousness or sedation.
 46. The method of claim 45 wherein the stepof contacting is done in vitro.
 47. The method of claim 45 wherein thestep of contacting is done in yeast cells containing recombinant formsof the first and second proteins.
 48. The method of claim 47 wherein thefirst and second recombinant proteins are each fused to a first andsecond yeast protein, wherein the first and second yeast proteinsreconstitute a functional transcriptional activator when brought intophysical proximity by binding of the first recombinant protein to thesecond recombinant protein.
 49. The method of claim 45 furthercomprising the step of: testing an identified candidate drug in ananimal to determine if the candidate drug relieves pain or inducesunconsciousness or sedation.
 50. The method of claim 45 wherein the testsubstance is contacted with PSD95 and nNOS.
 51. The method of claim 45wherein the test substance is contacted with PSD95 and NMDA receptor.52. The method of claim 45 wherein the test substance is contacted withPSD95, nNOS, and NMDA receptor.
 53. The method of claim 45 wherein thetest substance is contacted with PSD95 and NR2A.
 54. The method of claim45 wherein the test substance is contacted with PSD95 and NR2B.
 55. Themethod of claim 45 wherein the test substance is contacted with PSD93and nNOS.
 56. The method of claim 45 wherein the test substance iscontacted with PSD93 and NMDA receptor.
 57. The method of claim 45wherein the test substance is contacted with PSD93, nNOS, and NMDAreceptor.
 58. The method of claim 45 wherein the test substance iscontacted with PSD93 and NR2A.
 59. The method of claim 45 wherein thetest substance is contacted with PSD93 and NR2B.
 60. The method of claim45 wherein surface plasmon resonance is used to determine said amount.61. The method of claim 45 wherein antibodies are used to determine saidamount.
 62. The method of claim 13 wherein the anesthetic is aninhalational anesthetic.
 63. The method of claim 30 wherein theanesthetic is an inhalational anesthetic.
 64. The method of claim 13wherein the anesthetic is selected from the group consisting ofurethane, chloral hydrate, and sodium pentobarbitone.
 65. The method ofclaim 30 wherein the anesthetic is selected from the group consisting ofurethane, chloral hydrate, and sodium pentobarbitone.